Historically, traditional whole cell, growth-based assays have been the single most productive approach to the identification of anti-infectives that inhibit bacterial proliferation. None the less, growth-based assays have a number of important limitations including relatively low sensitivity/dynamic range relative to molecular assays, inability to distinguish bacteriostatic from bactericidal molecules, and poor performance in clinically important phases of bacterial disease, such as biofilms. To address these limitations, a number of improved cell-based screening approaches have been developed, each successful in its own right, but each also with an inherent set of limitations. For instance, Alamar blue dye, which measures the respiratory activity of organisms, is arguably the gold-standard whole cell antimicrobial screening technique. But, the dye cannot be used to effectively screen respiratory defective bacterial populations, such as small colony variants. Nor does the dye allow for distinction between bacteriostatic and bactericidal agents. We have developed a novel assay of antibacterial activity that directly detects the killing of bacteria by measuring the release of th intracellular enzyme adenylate kinase (AK) into the culture medium as a reporter of cell death. Our recent publication validated the use of the assay in antimicrobial screening and established that it has three key features. First, the assay is applicable to screening virtually any bacterial population. Second, the assay performs with exquisite sensitivity. Third, the assay enriches for the identification of bactericidal agents. As such, we believe the AK assay provides a very powerful new screening option for antimicrobial drug discovery, particularly for pathogens or bacterial growth states that are of immense healthcare concern that cannot be readily screened using conventional growth- based approaches and for which bactericidal antibiotics are desired; three examples include non-replicating (and slow growing) Mycobacterium tuberculosis, bacteria within established biofilms, and bacterial small colony variants. Accordingly, this application is being submitted in response to PA-10-213, Development of Assays for High Throughput Screening for use in Probe and Pre-therapeutic Discovery. Consistent with the goals of this program announcement, we propose to further develop and validate three AK-assay based high throughput screening strategies that would not be possible using growth based assays and for which bactericidal agents are needed. We will develop AK-based high throughput screens for agents that kill: 1) non-replicating (and slow growing) M. tuberculosis; 2) bacteria within established biofilms; and 3) bacterial small colony variants.